Macromolecules are involved in diverse and essential functions in living systems. The ability to decipher the functions, dynamics, and interactions of macromolecules is dependent upon an understanding of their chemical and three-dimensional structures. These three aspects—chemical and three-dimensional structures and dynamics—are interrelated. For example, the chemical composition of a protein, and more particularly the linear arrangement of amino acids, explicitly determines the three-dimensional structure into which the polypeptide chain folds after biosynthesis (Kim & Baldwin (1990) Ann. Rev. Biochem. 59: 631-660), which in turn determines the interactions that the protein will have with other macromolecules, and the relative mobilities of domains that allow the protein to function properly.
Biological macromolecules are either polymers or complexes of polymers. Different types of macromolecules are composed of different types of monomers, i.e., twenty amino acids in the case of proteins and four major nucleobases in the case of nucleic acids. A wealth of information can be obtained from a determination of the linear, or primary, sequence of the monomers in a polymer chain. For example, by determining the primary sequence of a nucleic acid, it is possible to determine the primary sequences of proteins encoded by the nucleic acid, to generate expression maps for the determination of mRNA expression patterns, to determine protein expression patterns, and to understand how mutations in genes correspond to a disease state. Furthermore, the characteristic pattern of distribution of specific nucleobase sequences along a particular DNA polymer can be used to unequivocally identify the DNA, as in forensic analysis. To this end, fast, accurate and inexpensive methods of characterizing polymers, and particularly nucleic acids, are being developed as a result of the endeavor of the Human Genome Project to sequence the human genome.
A challenge to the characterization of the linear sequence of monomers in a polymer chain has come from the natural tendency of polymers in most media to adopt unpredictable, coiled conformations. The average amount of such coiling is dependent on the interaction of the polymer with the surrounding solution, the rigidity of the polymer, and the energy of interaction of the polymer with itself. In most cases, the coiling is quite significant. For example, a λ-phage DNA, theoretically 16 μm long when stretched out so that the DNA is in the B conformation, has a random coil diameter of approximately 1 μm (Smith et al. (1989) Science 243:203-206).
DNA and many other biopolymers can be modeled as uniform elastic rods in a worm-like chain in order to determine their random coil properties (Austin et al. (1997) Physics Today 50(2):32-38). One relevant parameter is the persistence length, P, the length over which directionality is maintained, which is given by:P=κ/kBT  (1)where κ is the elastic bending modulus (Houseal et al. (1989) Biophys. J. 56:507-516), kB is the Boltzmann constant, and T is temperature (Austin et al. (1997) Physics Today 50(2):32-38). A longer persistence length means that the polymer is more rigid and more extended. Under physiological conditions, P=50 nm for DNA. While larger than the molecular diameter of 2.5 nm, the persistence length is many orders of magnitude smaller than the actual length of a typical DNA molecule such as a human chromosome, which is about 50 mm long. From the persistence length, the overall coil size, R, can be calculated (Austin et al. (1997) Physics Today 50(2):32-38) as follows:R2=2PL  (2)where L is the contour length of the DNA molecule. In the case of chromosomal DNA, R=70 μm. Clearly, it is much easier to analyze information on an extended piece of DNA that is 5 cm long than on a piece of DNA that has a coil size of 70 μm.
The force necessary to stretch polymers such as DNA is not very large. The worm-like chain model allows the polymer to be considered to be like a spring, and the force (Fs) needed to extend it close to its full natural length can be calculated (Austin et al. (1997) Physics Today 50(2):32-38) as follows:Fs≈kBT/P  (3)where all of the parameters are defined as above. Below Fs, the relationship between the force applied and the amount of stretching is roughly linear; above Fs, applying more force results in little change in the stretching (Smith et al. (1992) Science 258:1122-1126; Bustamante (1994) Science 265:1599-1600). Hence, full stretching is essentially attained by applying Fs. In the case of DNA, the force required to stretch it from its coiled conformation to its full length, which stretched conformation retains the B conformation is about 0.1 pN. Such a small force could, in principle, be obtained from virtually any source, including shear forces, electrical forces, and gravitational forces.
The danger in stretching DNA comes not in breaking the covalent bonds, which requires at least 1 nN of force (Grandbois et al. (1999) Science 283:1727-1730), but in over-stretching. It has been observed that, when 70 pN of force is applied, DNA adopts a super-relaxed form, called “S-DNA”, having nearly twice the length of normal B-form DNA having the same number of base pairs (Austin et al. (1997) Physics Today 50(2):32-38). Others have reported this transition at a force of 50 pN (Marko & Siggia (1995) Macromolecules 28:8759-8770). The length of S-DNA is less consistent than that of B-DNA stretched to its natural length and is more dependent on the exact force applied (Cluzel et al. (1996) Science 271:792-794), varying linearly with applied force from 1.7 to 2.1 times the length of B-DNA. Since it may not be possible to know the exact force applied, it is desirable to avoid stretching DNA into its S-form. Therefore, a force having a range of about two orders of magnitude, from about 0.1 pN to 25 pN, is capable of consistent and predictable stretching of DNA to its fully extended B-form.
In addition, the force must be applied fast enough to keep the polymer from recoiling. The natural relaxation time of a polymer, r, depends on the solvent, as follows (Marko (1998) Physical Review E 27:2134-2149):τ≈L2Pμ/kBT  (4)where μ is the viscosity of the solvent and the other parameters are as defined above. In the case of DNA at physiological conditions, the relaxation time is about 6 seconds, which can be increased to 20 seconds in a solution with a viscosity of 220 cp (Smith et al. (1999) Science 283:1724-1727) or by running the DNA in a confined space to lengthen P and change the viscous drag (Bakajin et al. (1998) Phys. Rev. Let. 80:2737-2740). Relaxation time is also a function of the extent of stretching (Hatfield & Quake (1999) Phys. Rev. Let. 82:3548-3551), so the values calculated above are a lower bound on the actual relaxation time.
Regardless of the exact value of the relaxation time, the polymer must be stretched out on a shorter time scale. In the case of flow through a channel, in which the stretching comes from fluid strain on the polymer, the appropriate time scale for stretching is the reciprocal of the strain rate. The strain rate is defined as dε/dt=dvX/dx, where x is the flow direction and vX is the x-component of the velocity. The multiple of the strain rate and the relaxation time is known as the Deborah number, De=τdε/dt, and can be used to determine whether the stretching will be maintained (Smith & Chu (1998) Science 281:1335-1340). If De is much greater than one, then the strain force predominates and the polymer will remain stretched. If De is much smaller than one, then the natural relaxation process dominates and the polymer will not remain stretched. When other stretching forces are involved, dimensionless values can be derived from other appropriate time scales, such as the Weissenberg number in extensional flow (Smith et al. (1999) Science 283:1724-1727).
Previous techniques used to stretch DNA involved immobilization of at least one end of the molecule on a surface, followed by manipulation of the other end, stretching with physical forces followed by immobilization, or running through a gel with constricted dimensions. Early attempts to stretch DNA for size measurement were conducted by Houseal et al. (1989, Biophys. J. 56:507-516). Contacting a DNA solution with a gold surface resulted in satisfactory binding, but use of the Kleinschmidt procedure, which is used extensively in electron microscopy to spread DNA molecules on a protein monolayer, resulted in a number of molecules remaining coiled instead of being stretched. Another attempt was made to stretch DNA by “gently” smearing it using a pipettor, a technique that is difficult to automate (PCT Publication No. WO 93/22463).
More sophisticated schemes have been devised for the immobilization of one end of DNA and other polymers on surfaces. In general, they involve the modification of a surface to expose reactive groups such as hydroxyl, amine, thiol, aldehyde, ketone, or carboxyl groups, or to attach such coupling structures as avidin, streptavidin, and biotin. Examples of these techniques are found in PCT Publication No. 97/06278; U.S. Pat. No. 5,846,724; and Zimmermann & Cox (1994) Nucl. Acids Res. 22:492-497. Often, these techniques involve the use of a silane (Bensimon et al. (1994) Science 265:2096-2098).
Once the polymer is immobilized on one end, stretching is possible since the forces may be aligned perpendicular to the attachment surface. One common method is to use a receding meniscus to align the polymer, a process sometimes referred to as “molecular combing.” In this technique, a second fluid is introduced that is substantially immiscible with the first, forming a meniscus at the interface. The original fluid is then gradually removed by mechanical, thermal, electrical, or chemical means or simply by evaporation and is replaced by the new fluid. As the interface moves, the polymer is aligned perpendicular to the interface by surface tension and therefore, becomes stretched. The force of stretching by this method is expressed as a function of the diameter D of the polymer (D=2.2 nm for double-stranded DNA) and the surface tension γ (Bensimon et al. (1994) Science 265:2096-2098): F=γπD.
With an air/water interface, γ is 0.07 N/m, giving a force of about 40 pN for DNA, which is clearly in the desired range. If the second fluid is properly chosen to discourage polymer movement, the polymer remains fixed in place indefinitely. Furthermore, adjacent polymers attached to the same surface all become aligned in the same direction. The two fluids involved, while often solvents of the polymer, can be only partial solvents and one can even be air. The degree of stretching is dependent on the modification of the surface (Bensimon, D. et al. (1995) Phys. Rev. Lett. 74(23):4754-4747), but is consistent for any given surface treatment. Variations of this technique have been employed (U.S. Pat. No. 5,851,769; PCT Publication No. WO 97/06278; Bensimon et al. (1994) Science 265:2096-2098; U.S. Pat. No. 5,840,862; Cox & Zimmermann (1994) Nucl. Acids Res. 22:492-497). Nevertheless, this technique cannot be easily adapted to a high-throughput operation, since the immobilization is a rate-limiting step and further modification of the polymer is more difficult after the immobilization.
An alternative way to manipulate DNA immobilized at one end involves the use of an optical trap. In this technique, a laser beam (“optical tweezers”) imparts momentum to a DNA molecule through emitted photons. By shifting the position of the photons, i.e., moving the beam, an extremely precise change can be induced in the direction of travel of the DNA (U.S. Pat. No. 5,079,169; Chu (1991) Science 253:861-866). Hence, a DNA molecule can be stretched using optical tweezers. The technique offers the advantage of being able to vary the force used for stretching and has been used to verify reptation theory (Perkins et al. (1994) Science 264:819-822). However, the laser can only hold one molecule in place at a time and has to be realigned for each subsequent molecule, making it unattractive for high-throughput analyses.
A third method of stretching DNA involves electrophoresis of either a DNA immobilized at one end to move the unattached end of the molecule away from the fixed end and subsequently attaching the fixed end to a surface with avidin, or a DNA unattached at both ends and then attaching both ends to a surface with avidin (Kabata et al. (1993) Science 262:1561-1563; Zimmerman & Cox (1994) Nucl. Acids Res. 22:492-497). No attempt was made to characterize the quality of the stretching using this technique. Furthermore, this technique shares the disadvantages of the previously-mentioned techniques (with respect to post-immobilization processing).
DNA has also been stretched by electrophoresis without fixing one end of the molecule. As part of a near-field detection scheme for sequencing biomolecules, DNA has been elongated by electrophoresis both in a gel and in solution, using electrical forces to move the DNA in position for reading (U.S. Pat. No. 5,538,898). However, no data were given to determine the quality of the stretching of large polymers, and the technique is limited to analyzing approximately 3 megabases at a time.
An extension of this idea involves the use of dielectrophoresis, or a field of alternating current, to stretch DNA. Washizu and Kurosawa ((1990) IEEE Transactions on Industry Applications 26:1165-1172) have demonstrated that DNA will stretch to its full length in its B-DNA form in a field having strength 106 V/m and a frequency of 400 kHz or more. At certain lower frequencies (around 10 kHz), the DNA will also stretch fully, but in a direction perpendicular to the field rather than parallel to it. This technology has been applied to sizing DNA by creating a gap with a tapered width between electrodes such that the DNA will align where the gap width equals the length of the DNA. It has also been found that this technique will not stretch single-stranded DNA due to differences in solvent interactions from double stranded DNA (Washizu et al. (1995) IEEE Transactions on Industry Applications 31:447-456). One disadvantage to this technique is that, due to the presence of induced dipoles along the length of the DNA, samples agglomerate readily, and in a heterogeneous sample, it is difficult to accurately identify the components. In addition, these experiments must be performed in deionized water in order to avoid the unwanted effects of Joule heating and electro-osmotic flow, presenting a sample preparation difficulty since most DNA exists in salt solutions or other solvents.
Gravitational forces have also been used to stretch DNA (U.S. Pat. No. 5,707,797; Windle (1993) Nature Genetics 5:17-21). In this technique, drops of DNA from the sodium dodecyl sulfate lysing of cells were allowed to run down a slide held at an angle. The effect of gravity was enough to stretch out the DNA, even to its over-stretched S-DNA form. The DNA was then immobilized on the slide, making processing, e.g., fluorescent labeling, prior to stretching relatively difficult.
Church et al. have developed another method for polymer characterization that involves measuring physical changes at an interface between two pools of media as a polymer traverses that interface (U.S. Pat. No. 5,795,782). This method is relatively inflexible. For example, the ion channel embodiment for nucleic acid characterization (Church et al. (1999) Science 284:1754-1756) works only for single stranded DNA. An interface usable for a wide variety of polymers has yet to be developed.
A method for measuring the length of DNA was developed by Kambara et al. (U.S. Pat. No. 5,356,776). This method involves electrophoresis of DNA through a gel; when the DNA reaches a portion of the gel no more than a few microns in diameter, it is forced into a straight line, where detection of fluorescent labels on each end of the DNA is accomplished. In another embodiment, the DNA is immobilized on one end in an aperture, stretched by electrophoresis, and a label on the other end of the molecule is detected. The use of a gel in this method necessitates a higher voltage than in solution to move DNA, and the end labeling precludes most other characterization of the DNA. In addition, long DNA molecules tend to become entangled in a gel. A modification of electrophoresis procedures, known as pulsed-field electrophoresis (Schwartz & Koval (1989) Nature 338:520-522), allows the full stretching of longer pieces of DNA by moving the electric field. However, this technique takes a substantially longer time to run because of the field variation and shares the other disadvantages of electrophoresis.
A hybrid of gel based and solution methods for stretching DNA was developed by Schwartz et al. ((1993) Science 262:110-113). DNA was placed in a free molten agarose solution, stretched by gravity, and then fixed in place by the gelling process. An enzyme was also added during gelling to cut the DNA at specific sites. This method is effective in creating restriction maps, however, predictable stretching in an agarose medium is difficult and the adaptation of the technique to high-throughput methods of analyzing uncut DNA is problematic.
Other techniques for characterizing particles do not rely on stretching. For example, a method developed by Schwartz (U.S. Pat. No. 5,599,664; EP 0391674) allows sizing and massing by subjecting a particle to a force and measuring conformational and positional changes. In the case of polymers, the force is usually applied to a coiled conformation. Another method for sizing and sorting DNA molecules (Chou et al. (1999) Proc. Natl. Acad. Sci. USA 96:11-13) involves a device that operates on a micron scale. The device uses the integral fluorescence signal from coiled DNA passing a detector to conduct the analysis. Schmalzing et al. ((1998) Analytical Chemistry 70:2303-2310; (1997) Proc. Natl. Acad. Sci. USA 94:10273-10278) developed microfabricated devices for DNA analysis, including sequencing which employ small-scale versions of traditional techniques, such as electrophoresis, and do not rely on DNA stretching.
In order to accurately determine the linear sequence of information in biopolymers, it is necessary to stretch the biopolymer so that individual units are distinguishable. Although many techniques have been developed that stretch biopolymers, and particularly DNA, they all have drawbacks, such as uniformity and reproducibility of stretching, ease of handling the biopolymer, and applicability to all types and sizes of biopolymers. Furthermore, none of them are applicable to rapid analysis of information, such as is necessary to sequence large pieces of DNA on a reasonable time scale. Clearly, there is a need for methods and apparatuses for reliably stretching polymers such that the linear sequence of information therein can be determined more rapidly and accurately in order to elucidate complex genetic function and diagnose diseases and genetic dysfunctions.
Citation of a reference herein shall not be construed as indicating that such reference is prior art to the present invention.